Abstract

Previous work carried out in our laboratory (Burlini, N., Lamponi S., Radrizzani, M., Monti, E. and Tortora P. (1987) Biochim. Biophys. Acta 930, 220-229) led to the immunological identification of a yeast 65-kDa phosphoprotein as a modified form of phosphoenolpyruvate carboxykinase; moreover the appearance of this phospho form was proven to be independent of cAMP, whereas the glucose-induced inactivation of the native enzyme is cAMP-dependent. Here, we report further investigations on the mechanism of the glucose-triggered degradation of the enzyme which led to the following results: (a) the aforementioned phospho form displayed a binding pattern to 5 AMP-Sepharose 4B quite similar to that of native enzyme, although it did not retain its oligomeric structure, nor was it catalytically active; (b) its phosphate content was of about two residues per monomer; (c) its isoelectric point was slightly higher than that of native enzyme, this shows that the enzyme undergoes additional modifications besides phosphorylation; (d) it represented about 4% of the native enzyme in glucose-depressed cells; (e) other forms immunologically cross-reactive with the native enzyme were also isolated, whose molecular mass was in the range of 60-62 kDa, and they are probable candidates as degradation products of the phospho form; (f) time courses of the native and phospho forms in the presence and the absence of glucose provided data consistent with a kinetic model involving a strong stimulation of the decay of both forms effected by the sugar; (g) in the mutant ABYS1 (Achstetter, T., Emter, O., Ehmann, C. and Wolf, D.H. (1984) J. Biol. Chem. 259, 13334-13343) which is devoid of the four major vacuolar proteinases, the decay pattern was essentially the same as in wild-type; (h) effectors lowering intracellular ATP also retarded the first step of enzyme degradation; this points to an ATP-dependence of this step. Based on these results we propose a degradation mechanism consisting of an initial cAMP- and ATP-dependent modification of the enzyme, followed by a cAMP-independent phosphorylation, which leads to the appearance of the aforementioned monomeric phospho form; this in turn seems to undergo limited proteolysis. These data strongly suggest the occurrence of an intermediate form arising from the native one and whose phosphorylation gives rise to the 65-kDa phosphoprotein described here.

Interaction Annotations

Increase the total number of rows showing on this page by using the pull-down located below the table, or use the
page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column
header to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small
"i" buttons located within a cell for an annotation to view further details about experiment type and any other
genes involved in the interaction.

Gene Ontology Annotations

Increase the total number of rows showing on this page using the pull-down located below the table, or use the page
scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header
to sort by that column; filter the table using the "Filter" box at the top of the table.

Phenotype Annotations

Increase the total number of rows showing on this page using the pull-down located below the table, or use the page
scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column header
to sort by that column; filter the table using the "Filter" box at the top of the table; click on the small "i"
buttons located within a cell for an annotation to view further details.

Regulation Annotations

Increase the total number of rows displayed on this page using the pull-down located below the table, or use the
page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column
header to sort by that column; to filter the table by a specific experiment type, type a keyword into the Filter box
(for example, “microarray”); download this table as a .txt file using the Download button or click Analyze to
further view and analyze the list of target genes using GO Term Finder, GO Slim Mapper, SPELL, or YeastMine.

Expression Datasets

Increase the total number of rows displayed on this page using the pull-down located below the table, or use the
page scroll at the table's top right to browse through the table's pages; use the arrows to the right of a column
header to sort by that column; filter the table using the "Filter" box at the top of the table; download this table
as a .txt file using the Download button;